Walking the Line: A Fibronectin Fiber-Guided Assay to Probe Early Steps of (Lymph)angiogenesis.

Angiogenesis and lymphangiogenesis are highly complex morphogenetic processes, central to many physiological and pathological conditions, including development, cancer metastasis, inflammation and wound healing. While it is described that extracellular matrix (ECM) fibers are involved in the spatiotemporal regulation of angiogenesis, current angiogenesis assays are not specifically designed to dissect and quantify the underlying molecular mechanisms of how the fibrillar nature of ECM regulates vessel sprouting. Even less is known about the role of the fibrillar ECM during the early stages of lymphangiogenesis. To address such questions, we introduced here an in vitro (lymph)angiogenesis assay, where we used microbeads coated with endothelial cells as simple sprouting sources and deposited them on single Fn fibers used as substrates to mimic fibrillar ECM. The fibers were deposited on a transparent substrate, suitable for live microscopic observation of the ensuing cell outgrowth events at the single cell level. Our proof-of-concept studies revealed that fibrillar Fn, compared to Fn-coated surfaces, provides far stronger sprouting and guidance cues to endothelial cells, independent of the tested mechanical strains of the Fn fibers. Additionally, we found that VEGF-A, but not VEGF-C, stimulates the collective outgrowth of lymphatic endothelial cells (LEC), while the collective outgrowth of blood vascular endothelial cells (HUVEC) was prominent even in the absence of these angiogenic factors. In addition to the findings presented here, the modularity of our assay allows for the use of different ECM or synthetic fibers as substrates, as well as of other cell types, thus expanding the range of applications in vascular biology and beyond.

Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis.

Lymphangiogenesis plays an important role in promoting cancer metastasis to sentinel lymph nodes and beyond and also promotes organ transplant rejection. We used human lymphatic endothelial cells to establish a reliable three-dimensional lymphangiogenic sprouting assay with automated image acquisition and analysis for inhibitor screening. This high-content phenotype-based assay quantifies sprouts by automated fluorescence microscopy and newly developed analysis software. We identified signaling pathways involved in lymphangiogenic sprouting by screening the Library of Pharmacologically Active Compounds (LOPAC)(1280) collection of pharmacologically relevant compounds. Hit characterization revealed that mitogen-activated protein kinase kinase (MEK) 1/2 inhibitors substantially block lymphangiogenesis in vitro and in vivo. Importantly, the drug class of statins, for the first time, emerged as potent inhibitors of lymphangiogenic sprouting in vitro and of corneal and cutaneous lymphangiogenesis in vivo. This effect was mediated by inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and subsequently the isoprenylation of Rac1. Supplementation with the enzymatic products of HMG-CoA reductase functionally rescued lymphangiogenic sprouting and the recruitment of Rac1 to the plasma membrane.

TScratch: a novel and simple software tool for automated analysis of monolayer wound healing assays.

Cell migration plays a major role in development, physiology, and disease, and is frequently evaluated in vitro by the monolayer wound healing assay. The assay analysis, however, is a time-consuming task that is often performed manually. In order to accelerate this analysis, we have developed TScratch, a new, freely available image analysis technique and associated software tool that uses the fast discrete curvelet transform to automate the measurement of the area occupied by cells in the images. This tool helps to significantly reduce the time needed for analysis and enables objective and reproducible quantification of assays. The software also offers a graphical user interface which allows easy inspection of analysis results and, if desired, manual modification of analysis parameters. The automated analysis was validated by comparing its results with manual-analysis results for a range of different cell lines. The comparisons demonstrate a close agreement for the vast majority of images that were examined and indicate that the present computational tool can reproduce statistically significant results in experiments with well-known cell migration inhibitors and enhancers.